Wide genetic diversity of Ralstonia solanacearum strains affecting tomato in Trinidad,West Indies

The genetic diversity of Ralstonia solanacearum causing bacterial wilt of tomato in Trinidad was assessed using the hierarchical phylotyping scheme and rep‐PCR DNA fingerprinting. Seventy‐one isolates were collected in 2003 on infected tomato crops in the four main vegetable cropping areas of Trinidad (North, Central, South‐East and South). Two phylotypes were present, with phylotype II being much more prevalent (66%) than phylotype I (34%). Phylotype II strains consisted mainly of sequevar 7 in Central and South‐East, and sequevar 35 in North, South‐East and South. This is the first report of sequevar 7 outside south‐eastern USA. In contrast, no ‘brown rot’ (phylotype IIB/1, race 3 biovar 2) or emerging strains of phylotype IIB/4NPB were identified. Rep‐PCR data were used to assess population genetic structure. No significant clustering by geographical distance was found, suggesting regular gene flow among cropping areas (via waterways, plant or soil). However, the population from Central was significantly differentiated from the others, containing only phylotype II/seq 7 strains, with a high degree of clonality, suggesting a possible recent introduction from abroad. The South population was less aggressive and more genetically diverse, suggesting horizontal gene transfers within the population, even among isolates of different phylotypes. Phylotype I and phylotype II populations differed slightly in clonality levels, with indications of more frequent recombination events within phylotype I populations. Possible factors influencing genetic diversity and distribution within the island are discussed.     

Host specificity and genetic diversity of race 4 strains of Ralstonia solanacearum

Ralstonia solanacearum race 4 isolates were obtained from Zingiberaceae plants in India during bacterial wilt outbreaks. Polyphasic phenotypic and genotypic analysis revealed intraracial diversity and dominance of biovar 3 over biovar 4. Biovar 3 strains were isolated from very severely wilted Zingiberaceae plants in the field and found to be present across diverse geographical, host and seasonal boundaries. It was hypothesized that these isolates belong to a single, ‘fast wilting’, lineage. Using one ‘fast wilting’ isolate in controlled inoculations, rapid wilt was observed in ginger within 5–7 days. Wilting was also observed in several other closely and distantly related hosts such as turmeric (Curcuma longa), aromatic turmeric (Curcuma aromatica), black turmeric (Curcuma caesia), sand ginger (Kaempferia galanga), white turmeric (Curcuma zeodaria), awapuhi (Zingiber zerumbet), greater galangal (Alpinia galanga), globba (Globba sp.), small cardamom (Elettaria cardamomum) and large cardamom (Ammomum subulatum) of the Zingiberaceae family, and in tomato (Solanum lycopersicum). Molecular analysis, including multiplex PCR‐based phylotyping, sequence analysis of 16S rDNA, 16–23S intergenic spacer and the recN gene, and multilocus sequence typing, revealed minimal differences between fast wilting isolates, confirming that almost all belong to the same lineage. Biovar 4 was isolated from plants showing slow wilt progression and self‐limiting wilting in restricted geographical locations instead, and was identified to be genetically distinct from the fast wilting biovar 3 isolates. To the authors' knowledge, this is the first report of host range and genetic analysis of R. solanacearum race 4 in India.     

番茄青枯病内生拮抗细菌的筛选

 从广西一些市县采集番茄茎标本分离得到55个细菌菌株,分属为芽孢杆菌(Bacillus spp.)、黄单胞菌(Xanthomonas spp.)、假单胞菌(Pseudomonas spp.)和欧文氏菌(Erwinia spp.),其中芽孢杆菌为优势种群。经回接测试,有36个菌株为番茄植株内生菌。这些内生菌只有7个菌株对番茄青枯病菌有拮抗作用,芽孢杆菌B₄₇菌株对番茄青枯病菌拮抗作用较强,经室内和田间初步防治测定,它对番茄青枯病有较好的防治效果。     

Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurrence of tomato fruit infection

Fifty-nine Ralstonia solanacearum isolates from diverse crops and regions were collected and characterized to determine the distribution and diversity of this soilborne pathogen in Guatemala. Three distinct types were present: a phylotype I, sequevar 14 strain, probably originating from Asia, infecting tomatoes and aubergines at moderate elevations; a phylotype II, sequevar 6 strain of American origin causing Moko disease in lowland banana plantations; and a phylotype II, sequevar 1 (race 3 biovar 2) strain causing brown rot on potatoes, Southern wilt of Pelargonium spp. and bacterial wilt of greenhouse tomatoes at high elevations. These data on strain diversity will inform effective regional efforts to breed for wilt resistance. A sensitive enrichment method did not detect the pathogen in fruits from naturally infected commercial tomato plants in Guatemalan fields and greenhouses, although it was detected in 6% of fruits from a wilt-resistant hybrid. Low numbers of R. solanacearum cells were also infrequently detected in fruits from plants artificially inoculated in the growth chamber with either race 3 biovar 2 or a phylotype II tomato strain.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

Assessment of resistance to bacterial wilt incited by Ralstonia solanacearum in tomato germplasm

Journal of Plant Diseases and Protection - Bacterial wilt incited by Ralstonia solanacearum has been found the most damaging and widespread diseases of tomato throughout the world and causes heavy...     

Ralstonia solanacearum virulence in tomato seedlings inoculated by leaf clipping

Ralstonia solanacearum is a phytopathogenic bacterium that colonizes the xylem vessels of host plants leading to a lethal wilt disease. Although several studies have investigated the virulence of R. solanacearum on adult host plants, infection studies of this pathogen on the seedling stages of hosts are less common. In a preliminary observation, inoculation of R. solanacearum F1C1 on 6‐ to 7‐day‐old tomato seedlings by a simple leaf‐clip strategy resulted in a lethal pathogenic condition in seedlings that eventually killed these seedlings within a week post‐inoculation. This prompted testing of the effect of this inoculation technique in seedlings from different cultivars of tomato and similar results were obtained. Colonization and spread of the bacteria throughout the infected seedlings was demonstrated using gus‐tagged R. solanacearum F1C1. The same method of inoculating tomato seedlings was used with R. solanacearum GMI1000 and independent mutants of R. solanacearum GMI1000, deficient in the virulence genes hrpB, hrpG, phcA and gspD. Wildtype R. solanacearum GMI1000 was found to be virulent on tomato seedlings, whereas the mutants were found to be non‐virulent. This leaf‐clip technique, for inoculation of tomato seedlings, has the potential to be a valuable approach, saving time, space, labour and costs.     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

Bacterial wilt of bellflower caused by Ralstonia solanacearum in Japan

A sudden wilt of bellflower (Campanula lactiflora) was observed in Japan in 1997. A bacterium that formed white fluidal and mucoid colonies resembling those of Ralstonia solanacearum was isolated from the infected plants. The bacterium was bacteriologically identified as biovar 3 of R. solanacearum. This is the first report of R. solanacearum affecting a plant species of the Campanulaceae family.     

Resistance mechanisms of wild tomato germplasm to infection of Oidium neolycopersici

Tomato powdery mildew (Oidium neolycopersici) is one of the most devastating diseases of cultivated tomatoes worldwide. Although the first epidemics were recorded more than 25 years ago many aspects of this host-pathogen interaction are still not well understood. Detailed morphological and molecular studies of the anamorphs confirmed that O. neolycopersici is phylogeneticaly close to Erysiphe aquilegiae var. ranunculi. Host range is rather broad, apart from Solanaceae hosts were found in the families Apocynaceae, Campanulaceae, Crassulaceae, Cistaceae, Cucurbitaceae, Linaceae, Malvaceae, Papaveraceae, Pedialiaceae, Scrophulariaceae, Valerianaceae a Violaceae. Non-host resistance within these families is not based on inhibition of formation of primary haustorium, however, on post-haustorial hypersensitive reponse and another type of non-hypersensitive resistance. Screening of wild Solanum species (previous Lycopersicon spp.) germplasm revealed valuable sources of resistance (S. habrochaites, S. pennellii, S. cheesmaniae, S. chilense, S. peruvianum). The main resistance mechanism was found to be a hypersensitive response (HR), in some cases followed by limited development of the pathogen. However, there is a broad variation in resistance response on the histological and cytological level. Interaction between many wild Solanum spp. and O. neolycopersici is race-specific, at least three races were differentiated. In some interspecific crosses (S. lycopersicum × S. habrochaites) adult plant resistance was observed. Biochemical studies focusing on production of reactive oxygen species (ROS) and peroxidase activity during infection of O. neolycopersici showed that production of ROS and activity of corresponding enzymes is related to activation of defence responses in genotypes of wild Solanum sect. Lycopersicon. The significance of nitric oxide (NO) in O. neolycopersici pathogenesis was supported by experiments with NO donors and scavengers. In moderately resistant genotype S. chmielewskii, treatment by heat stress caused slight deceleration of pathogen development, increased production of jasmonic acid (JA) and abscisic acid (ABA) and increased peroxidase activity in infected plants. The different degree of tomato resistance/susceptibility did not markedly change the rate and extent of photosynthetic response to O. neolycopersici; only minimal impairment of photosynthesis was found in both susceptible and moderately resistant genotypes during the first 9 days after inoculation. The accumulated evidence confirm a crucial role of localised increased production of ROS and reactive nitrogen species (RNS) in response to pathogen penetration into plant tissue and its involvement in the plant resistance responses including the initiation and progression of plant cell death in host wild Solanum species. Crucial points of further research are discussed.     

Phylogenetic and pathogenic variability of strains of Ralstonia solanacearum causing moko disease in Colombia

Moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most devastating diseases of Musa spp. in Colombia, where banana and plantain are major crops. The disease epidemiology is poorly understood and little is known about the diversity of the bacterial populations associated with this disease. This study assessed the diversity, phylogenetic relationship and pathogenicity of R. solanacearum strains associated with moko disease in Colombia. For this, the genetic diversity of 65 isolates obtained from four banana/plantain-growing regions was evaluated by using multiplex PCR and analysing the partial sequences of the mutS, rplB and egl genes. These analyses revealed that all the strains belonged to the R. solanacearum phylotype II, sequevars 4 and 6. In addition, the phylogenetic analysis assorted the strains into three subgroups, which matched the region of isolation: (i) central region (i.e. Eastern plains and Andes, IIB/4); (ii) northwest (i.e. Urabá and a few strains from Magdalena, IIB/4); and (iii) north coast (Magdalena and a few strains from Urabá, IIA/6). In addition, this evolutionary pattern was associated with pathogenicity, as 63 of the 65 isolates caused wilting of banana and plantain plants under greenhouse conditions, whilst only 32, those isolated from the central region, caused such symptoms in tomato plants. In conclusion, this study shows that banana and plantain crops in Colombia foster genetically diverse strains of R. solanacearum that belong to at least three different genetic groups, which show biogeographic and host range association.     

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757     

青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析

 利用青枯雷尔氏菌(Ralstonia solanacearum)无致病力菌株防治番茄青枯病具有很好的应用潜力。作者通过分离筛选自然弱毒株、⁶⁰Co辐射诱变和EZ-Tn5插入诱变,分别获得3、12和40株青枯雷尔氏菌无致病力突变菌株。经盆栽番茄苗致病性检测,15 d后均未发病,证实均为无致病力青枯雷尔氏菌。进一步对番茄青枯病的防治试验表明,从番茄青枯病发病田块分离的无致病力突变菌株FJAT1458的防治效果最好,防效达100%。该菌株能定殖番茄植株根系土壤、根部和茎部,定殖数量均表现为“先增后减”的趋势,并且接种浓度越大、苗龄越小,定殖数量越大。从构建的防效模型可以看出,不同接种浓度条件下,植株发病率随时间变化符合的回归方程不同,相关系数R值也不同,接种浓度越大,R值越小。本研究获得的青枯雷尔氏菌无致病力突变菌株FJAT1458对番茄青枯病具有很好的防病效果。     

Implication of C-terminal mutation of PopA of Ralstonia solanacearum strain OE1-1 in suppression of bacterial wilt

Ralstonia solanacearum strain OE1-1 causes bacterial wilt on tobacco plants. The popA -mutant 31b, derived from OE1-1 by insertion of transposon Tn 4431 , did not cause wilt on tobacco plants inoculated through the roots. However, when 31b was directly inoculated into xylem vessels, the tobacco plants wilted, similarly to those inoculated with OE1-1. 31b retained its exopolysaccharide productivity and its type-III secretion function. Furthermore, 31b grew in intercellular spaces and systemically infected tobacco plants, similarly to OE1-1. popA consists of an operon with popB and popC , and suppression of popB and popC expression resulting from polar mutation by transposon insertion did not affect the virulence of 31b. The mutated popA ( popA31b ) was composed of 960 nucleotides, including 39 derived from Tn 4431. A recombinant mutant from OE1-1, where popA31b was introduced by marker exchange, showed the same phenotype as 31b. PopA31b protein was extracellularly secreted by 31b co-cultured with Arabidopsis thaliana . These results suggest that PopA31b extracellularly secreted by 31b in intercellular spaces may be implicated in suppression of disease development, leading to inability of the bacteria to induce wilt on plants. Taken together, interactions between host plants and R. solanacearum existing in intercellular spaces immediately after invasion may be involved in disease development.     

Involvement of HLK effectors in Ralstonia solanacearum disease development in tomato

A multigene family encoding HLK effectors is well conserved in all Ralstonia solanacearum strains. The HLK family contains three paralogous members. We constructed single, double, and triple deletion mutants of hlk genes in R. solanacearum strain OE1-1 and characterized HLK effectors by comparing the phenotypes of the strains. HLK effectors exhibited some functional redundancy. While the triple deletion mutant was significantly impaired in virulence on tomato and multiplied less efficiently than the wild type in the leaves, the mutant was as virulent as the wild type on eggplant and tobacco.     

林木青枯病研究进展

青枯病是由青枯劳尔氏菌引起的一种严重的植物土传病害,我国南方多种树种被其侵染发病。本文介绍了我国林木青枯病的发生情况,概括了青枯病检测与防治方面的研究进展,并对其中的一些问题进行了探讨。     

Identification and use of a wild plant with antimicrobial activity against Ralstonia solanacearum, the cause of bacterial wilt of potato

Fresh aerial tissue and roots of 14 wild plants in Okinawa prefecture were investigated for their antimicrobial activity against Ralstonia solanacearum , which causes bacterial wilt of potato. A 70% aqueous ethanol extract of fresh aerial tissue of Geranium carolinianum L. showed strong antimicrobial activity against R. solanacearum . This extract also showed antimicrobial activity against the pathogens causing common scab of potato and soil rot of sweet potato. The antimicrobial substance could be extracted with hot water, and was effective against R. solanacearum in soil. In the field test, a treatment combining incorporation of dried aerial tissue into the soil and solarization was highly effective for control of bacterial wilt of potato. These findings suggest that G. carolinianum L. could be used as a biological agent for the control of bacterial wilt of potato.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050